Engineering the Staple Oil Crop Brassica napus Enriched with α-Linolenic Acid Using the Perilla FAD2-FAD3 Fusion Gene.

Modern people generally suffer from α-linolenic acid (ALA) deficiency, since most staple food oils are low in ALA content. Thus, the enhancement of ALA in staple oil crops is of importance. In this study, the FAD2 and FAD3 coding regions from the ALA-king species Perilla frutescens were fused using a newly designed double linker LP4-2A, driven by a seed-specific promoter PNAP, and engineered into a rapeseed elite cultivar ZS10 with canola quality background. The mean ALA content in the seed oil of PNAP:PfFAD2-PfFAD3 (N23) T5 lines was 3.34-fold that of the control (32.08 vs 9.59%), with the best line being up to 37.47%. There are no significant side effects of the engineered constructs on the background traits including oil content. In fatty acid biosynthesis pathways, the expression levels of structural genes as well as regulatory genes were significantly upregulated in N23 lines. On the other hand, the expression levels of genes encoding the positive regulators of flavonoid-proanthocyanidin biosynthesis but negative regulators of oil accumulation were significantly downregulated. Surprisingly, the ALA level in PfFAD2-PfFAD3 transgenic rapeseed lines driven by the constitutive promoter PD35S was not increased or even showed a slight decrease due to the lower level of foreign gene expression and downregulation of the endogenous orthologous genes BnFAD2 and BnFAD3.

Development of a CRISPR-SaCas9 system for projection- and function-specific gene editing in the rat brain.

A genome editing technique based on the clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease Cas9 enables efficient modification of genes in various cell types, including neurons. However, neuronal ensembles even in the same brain region are not anatomically or functionally uniform but divide into distinct subpopulations. Such heterogeneity requires gene editing in specific neuronal populations. We developed a CRISPR-SaCas9 system-based technique, and its combined application with anterograde/retrograde AAV vectors and activity-dependent cell-labeling techniques achieved projection- and function-specific gene editing in the rat brain. As a proof-of-principle application, we knocked down the cbp (CREB-binding protein), a sample target gene, in specific neuronal subpopulations in the medial prefrontal cortex, and demonstrated the significance of the projection- and function-specific CRISPR-SaCas9 system in revealing neuronal and circuit basis of memory. The high efficiency and specificity of our projection- and function-specific CRISPR-SaCas9 system could be widely applied in neural circuitry studies.

Shp-1 dephosphorylates TRPV1 in dorsal root ganglion neurons and alleviates CFA-induced inflammatory pain in rats.

Transient receptor potential vanilloid 1 (TRPV1) receptors are expressed in nociceptive neurons of rat dorsal root ganglions (DRGs) and mediate inflammatory pain. Nonspecific inhibition of protein-tyrosine phosphatases (PTPs) increases the tyrosine phosphorylation of TRPV1 and sensitizes TRPV1. However, less is known about tyrosine phosphorylation's implication in inflammatory pain, compared with that of serine/threonine phosphorylation. Src homology 2 domain-containing tyrosine phosphatase 1 (Shp-1) is a key phosphatase dephosphorylating TRPV1. In this study, we reported that Shp-1 colocalized with and bound to TRPV1 in nociceptive DRG neurons. Shp-1 inhibitors, including sodium stibogluconate and PTP inhibitor III, sensitized TRPV1 in cultured DRG neurons. In naive rats, intrathecal injection of Shp-1 inhibitors increased both TRPV1 and tyrosine-phosphorylated TRPV1 in DRGs and induced thermal hyperalgesia, which was abolished by pretreatment with TRPV1 antagonists capsazepine, BCTC, or AMG9810. Complete Freund's adjuvant (CFA)-induced inflammatory pain in rats significantly increased the expression of Shp-1, TRPV1, and tyrosine-phosphorylated TRPV1, as well as the colocalization of Shp-1 and TRPV1 in DRGs. Intrathecal injection of sodium stibogluconate aggravated CFA-induced inflammatory pain, whereas Shp-1 overexpression in DRG neurons alleviated it. These results suggested that Shp-1 dephosphorylated and inhibited TRPV1 in DRG neurons, contributing to maintain thermal nociceptive thresholds in normal rats, and as a compensatory mechanism, Shp-1 increased in DRGs of rats with CFA-induced inflammatory pain, which was involved in protecting against excessive thermal hyperalgesia.

Lysine-specific demethylase 1 in breast cancer cells contributes to the production of endogenous formaldehyde in the metastatic bone cancer pain model of rats.

BACKGROUND: Bone cancer pain seriously affects the quality of life of cancer patients. Our previous study found that endogenous formaldehyde was produced by cancer cells metastasized into bone marrows and played an important role in bone cancer pain. However, the mechanism of production of this endogenous formaldehyde by metastatic cancer cells was unknown in bone cancer pain rats. Lysine-specific demethylase 1 (LSD1) is one of the major enzymes catalyzing the production of formaldehyde. The expression of LSD1 and the concentration of formaldehyde were up-regulated in many high-risk tumors. OBJECTIVE: This study aimed to investigate whether LSD1 in metastasized MRMT-1 breast cancer cells in bone marrows participated in the production of endogenous formaldehyde in bone cancer pain rats. METHODOLOGY/PRINCIPAL FINDINGS: Concentration of the endogenous formaldehyde was measured by high performance liquid chromatography (HPLC). Endogenous formaldehyde dramatically increased in cultured MRMT-1 breast cancer cells in vitro, in bone marrows and sera of bone cancer pain rats, in tumor tissues and sera of MRMT-1 subcutaneous vaccination model rats in vivo. Formaldehyde at a concentration as low as the above measured (3 mM) induced pain behaviors in normal rats. The expression of LSD1 which mainly located in nuclei of cancer cells significantly increased in bone marrows of bone cancer pain rats from 14 d to 21 d after inoculation. Furthermore, inhibition of LSD1 decreased the production of formaldehyde in MRMT-1 cells in vitro. Intraperitoneal injection of LSD1 inhibitor pargyline from 3 d to 14 d after inoculation of MRMT-1 cancer cells reduced bone cancer pain behaviors. CONCLUSION: Our data in the present study, combing our previous report, suggested that in the endogenous formaldehyde-induced pain in bone cancer pain rats, LSD1 in metastasized cancer cells contributed to the production of the endogenous formaldehyde.

Contribution of the spinal cord BDNF to the development of neuropathic pain by activation of the NR2B-containing NMDA receptors in rats with spinal nerve ligation.

The NMDA receptor and the brain-derived neurotrophic factor (BDNF) are involved in central sensitization and synaptic plasticity in the spinal cord. To determine whether the spinal cord BDNF contributes to the development and maintenance of neuropathic pain by activation of the dorsal horn NR2B-containing NMDA (NMDA-2B) receptors, this study was designed to investigate if alterations in BDNF and its TrkB receptor in the spinal dorsal horn would parallel the timeline of the development of neuropathic pain in lumbar 5 (L5) spinal nerve ligated (SNL) rats. The enzyme-linked immunosorbent assay (ELISA) showed that the BDNF concentration significantly increased during 24 h post-surgery, and the maximal enhancement lasted for 48 h. It declined as time progressed and returned to the level of pre-operation at 28 days after SNL. In parallel with the alteration of BDNF concentration in the spinal dorsal horn, the 50% paw withdrawal threshold (PWT) of the ipsilateral hind paw in SNL rats also showed a significant decrease during 24-48 h after SNL as compared with those in sham-operated rats. The correlation analysis revealed that the BDNF concentration had a negative correlation with 50% PWT in early stage (0-48 h) (r=-0.974, p=0.001), but not late stage (3-28 days) (r=0.3395, p=0.6605), after SNL. Similarly, the immunohistochemical staining revealed that a significant up-regulation of BDNF expression in the spinal dorsal horn appeared as early as 12 h post-operation in SNL rats, peaked at 24-48 h, declined at 3 days and disappeared at 14 days after SNL. In contrast, an increase in NMDA-2B receptors expression in the spinal dorsal horn was delayed to 48 h after SNL. The increase reached peak at 3 days, lasted for 14 days, and returned to the control level of pre-operation at 28 days after SNL. The maximal enhancement of BDNF expression occurred in early stage (24-48 h) after nerve injury, while the peak of NMDA-2B receptors expression appeared in late stage (3-14 days) post-nerve ligation. As compared with the dynamic changes of 50% PWT in the timeline after nerve injury, the maximal enhancement of BDNF expression closely paralleled the maximal decline in the slope of 50% PWT, while the peak of NMDA-2B receptors expression corresponded with the plateau of the decreased 50% PWT. Therefore, the increased BDNF in the spinal dorsal horn was likely to be associated with the initiation of neuropathic pain in early stage (0-48 h), while the activation of NMDA-2B receptors was involved in the maintenance of persistent pain states in late stage (2-14 days) after nerve injury. Moreover, the present study also demonstrated that the BDNF/TrkB-mediated signaling pathway within the spinal cord might be involved in the induction of neuropathic pain in early stage after nerve injury, and the selective NMDA-2B receptors antagonist (Ro 25-6981) almost completely blocked the BDNF-induced mechanical allodynia in all of the tested rats. These data suggested that the BDNF/TrkB-mediated signaling pathway in the spinal cord was involved in the development of nerve injury-induced neuropathic pain through the activation of dorsal horn NMDA-2B receptors.